The pathogenesis and exacerbation of many prevalent T-cell mediated diseases result from an inappropriate immune response driven by abnormal T-cell activation. A number of other diseases are thought to be caused by aberrant T-cell activation including Type I (insulin-dependent) diabetes mellitus, thyroiditis, sarcoidosis, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (Crohn's and ulcerative colitis) and aplastic anemia. In addition, a variety of syndromes including septic shock and tumor-induced cachexia may involve T-cell activation and augmented production of potentially toxic levels of lymphokines. Normal T-cell activation also mediates the rejection of transplanted cells and organs by providing the neccessary signals for the effective destruction of the “foreign” donor tissue.
The activation of T-lymphocytes leading to T-cell proliferation and gene expression and secretion of specific immunomodulatory cytokines requires two independent signals. The first signal involves the recognition, by specific T-cell receptor/CD3 complex, of antigen presented by major histocompatibility complex molecules on the surface of antigen-presenting cells (APCs). Antigen-nonspecific intercellular interactions between T-cells and APCs provide the second signal which serves to regulate T-cell responses to antigen. These secondary or costimulatory signals determine the magnitude of a T-cell response to antigen. Costimulated cells react by increasing the levels of specific cytokine gene transcription and by stabilizing selected mRNAs. T-cell activation in the absence of costimulation results in an aborted or anergic T-cell response. One key costimulatory signal is provided by interaction of the T-cell surface receptor CD28 with B7-related molecules on APC (Linsley and Ledbetter (1993) Annu Rev Immunol 11: 191–212). CD28 is constitutively expressed on 95% of CD4+ T-cells (which provide helper functions for B-cell antibody production) and 50% of CD8+ T-cells (which have cytotoxic functions) (Yamada et al (1985) Eur J Immunol 15: 1164–1168). Following antigenic or in vitro mitogenic stimulation, further induction of surface levels of CD28 occurs, as well as the production of certain immunomodulatory cytokines. These include interleukin-2 (IL-2), required for cell cycle progression of T-cells, interferon-gamma (IFNγ), which displays a wide variety of anti-viral and anti-tumor effects and interleukin-8 (IL-8), known as a potent chemotactic factor for neutrophils and lymphocytes. These cytokines have been shown to be regulated by the CD28 pathway of T-cell activation (Fraser et al (1991) Science 251: 313–316, Seder et al (1994) J Exp Med 179: 299–304, Wechsler et al (1994) J Immunol 153: 2515–2523). IL-2, IFNγ and IL-8 are essential in promoting a wide range of immune responses and have been shown to be overexpressed in many T-cell mediated disease states.
In psoriasis, activated lesional T-cells predominantly release Th1 cytokines such as IL-2 and IFNγ (Schlaak et al (1994) J Invest Derm 102: 145–149). These secreted cytokines induce normal keratinocytes to express the same phenotype (HLA DR+/ICAM-1+) as found in psoriasis lesions (Baadsgaard et al (1990) J Invest Derm 95: 275–282). Also IL-8, by virtue of its in vitro and in vivo proinflammatory properties and because it is secreted in large amounts by both activated T-cells and keratinocytes from psoriatic lesions, is considered a major contributor to the pathologic changes seen in psoriatic skin such as keratinocyte hyperproliferation. Furthermore, one of the B7 family of receptors (the natural ligands for CD28 found on activated APC), BB1 has been shown to be expressed in psoriatic but not unaffected skin keratinocytes (Nickoloff et al (1993) Am J Pathology 142: 1029–1040) underscoring the importance of T-cell activation in pathogenesis of the disease.
In other T-cell mediated skin disorders such as allergic contact dermatitis and lichen planus, CD28 was expressed in high levels in the majority of dermal and epidermal CD3+ T-cells, but in normal skin and basal cell carcinoma (a non T-cell mediated skin disease), CD28 was expressed only in perivascular T-cells. Similarly, in both allergic contact dermatitis and lichen planus, B7 expression was found on dermal dendritic cells, dermal APCs and on keratinocytes, but not in normal skin and basal cell carcinoma (Simon et al (1994) J Invest Derm 103: 539–543). Therefore this suggests that the CD28/B7 pathway is an important mediator of T-cell-mediated skin diseases.
Aberrant T-cell activation associated with certain autoimmune diseases caused by the loss of self-tolerance is predominantly characterized by the presence of CD28+T-cells and expression of its ligand, B7 on activated professional APCs (monocyte, macrophage or dendritic cells). These include autoimmune Graves thyroiditis (Garcia-Cozar et al (1993) Immunologia 12 32), sarcoidosis (Vandenberghe et al (1993) Int Immunol 5: 317–321), rheumatoid arthritis (Verwilghen et al (1994) J Immunol 153: 1378–1385) and systemic lupus erythematosus (Sfikakis et al (1994) Clin Exp Immunol 96: 8–14). In normal T-cell activation, which mediates the rejection of transplanted cells and organs, the binding of CD28 by its appropriate B7 ligand during T-cell receptor engagement is critical for proper allogeneic response to foreign antigens, for example, on donor tissue (Azuma et al (1992) J Exp Med 175: 353–360, Turka et al (1992) Proc Nat Acad Sci USA 89: 11102–11105).
Traditional therapies for autoimmune diseases do not prevent T-cell activation; the effector step in the autoreactive immune responses to self-antigen. Drugs, such as steroids and non-steroid anti-inflammatory drugs (NSAIDS), are currently used to ameliorate symptoms, but they do not prevent the progression of the disease. In addition, steroids can have side effects such as inducing osteoporosis, organ toxicity and diabetes, and can accelerate the cartilage degeneration process and cause so-called post-injection flares for up to 2 to 8 hours. NSAIDS can have gastrointestinal side effects and increase the risk of agranulocytosis and iatrogenic hepatitis.
Immunosuppressive drugs are also used as another form of therapy, especially in advanced disease stages. However, these drugs suppress the entire immune system and often treatment has severe side effects including hypertension and nephrotoxicity. Also established immunosuppressants such as cyclosporin and FK506 cannot inhibit the CD28-dependent T-cell activation pathway (June et al (1987) Mol Cell Biol 7: 4472–4481).
Current agents which affect T-cell activation include synthetic peptides, monoclonal antibodies and soluble forms of T-cell activation molecules. To date competitive synthetic peptides to T-cell activation molecules such as CD28, CD40L and the CAM family of adhesion molecules have not been identified. Monoclonal antibodies (mAb) have been shown to have possible therapeutic effect in such T-cell mediated diseases such as psoriasis (anti-CD4 (Prinz et al (1994) Lancet 338: 320–321)) and immununosuppression of normal T-cell activation in allografts (anti-VCAM-1 and VLA-4 (Isobe et al (1994) J Immunol 153: 5810–5818)). However, with chronic treatment, the host animal develops antibodies against the monoclonal antibodies thereby limiting their usefulness. ‘Humanized’ monoclonal antibodies, have been developed which apparently reduce the risk of an induced immune response to these mAbs. However, these are still under development and in addition, these new mAbs remain large proteins and therefore may have difficulty reaching their target sites. Soluble forms of T-cell activation molecules such as CTLA-4Ig, containing the extracellular domain of the human CTLA-4 gene (which is sequentially related to CD28), fused to a human Ig Cγ chain, have been developed. CTLA-4Ig has been shown to specifically block normal T-cell activation by preventing rejection of xenogeneic (Lenschow et al (1992) Science 257: 789–792) and allogeneic (Turka et al (1992) Proc Nat Acad Sci USA 89: 11102–11105) cardiac allografts in rats and have therapeutic effect on aberrant T-cell activation such as found in rat autoimmune glomerulonephritis (Nishikawa et al (1994) Eur J Immunol 24: 1249–1254). Soluble CTLA-4Ig however suffers from similar limitations as monoclonal antibodies in addition to the expense of their production. Also the true function of this CD28-like molecule is not known therefore this needs to be fully determined before any therapeutic benefit can be evaluated.
Inhibition of the cell-surface expression of CD28 leads to prolonged unresponsiveness or deletion of activated T-cells. Inactivation prevents T-cell proliferation and arrest of T-cell-specific production of specific immunoregulatory cytokines such as interleukin-2, interferon-gamma and interleukin-8.
Regulation of CD28 gene expression can be achieved using antisense and triplex-forming oligonucleotides by hybridizing oligodeoxy-ribonucleotides or oligoribonucleotides to DNA or RNA sequences within the CD28 gene or promoter region (See PCT/US96/01507, filed Aug. 30, 1996). Oligonucleotides avoid many of the pitfalls of current agents used to block the effects of normal and abnormal T-cell activation. However, these oligos designed for antisense strategies are susceptible to degradation by intracellular nucleases or nucleases present in the extracellular milieu.
The binding of DNA (or RNA) to protein has been shown previously to be a fundamental pathway by which transcription of a gene is controlled. These regulatory proteins or transcription factors recognize DNA sequences with specific secondary structure and the ensuing interaction can lead to positive or negative control of gene expression. Aptamers are short oligonucleotide sequences which can specifically bind specific proteins. It has been demonstrated that different aptameric sequences can bind specifically to different proteins, for example, the sequence GGNNGG where N=guanosine (G), cytosine (C), adenosine (A) or thymidine (T) binds specifically to thrombin (Bock et al (1992) Nature 355: 564–566 and U.S. Pat. No. 5,582,981 (1996) Toole et al).
Aptameric sequences have not been described, however, which can function as competitive inhibitors of DNA-binding sites on regulatory proteins known as transcription factors. Transcription factors are a class of proteins which regulate genes by primarily binding to specific regulatory sequences in the 5′ upstream promoter region of those genes. This interaction leads to initiation of transcription. Certain transcription factors such as Sp1, AP2, AP-1, EGR-1 and NFκB are critical in the activation of T and B lymphocytes (Skerka et al J Biol Chem 270: 22500–22506, Jung et al (1995) Ann N Y Acad Sci 766: 245–252). In some cases these transcription factors are induced by signals initiated following costimulation (Jung et al (1995) Ann N Y Acad Sci 766: 245–252). Thus, there is still a need to develop agents and methods for interfering with the interaction of protein with specific DNA binding sites which would lead to suppression of certain immune pathways including the costimulatory pathway.